Primed to Die: An Investigation of the Genetic Mechanisms Underlying Noise-Induced Hearing Loss and Cochlear Damage in Homozygous Foxo3-knockout Mice

Abstract

The prevalence of noise-induced hearing loss (NIHL) continues to increase, with limited therapies available for individuals with cochlear damage. We have previously established that the transcription factor FOXO3 is necessary to preserve outer hair cells (OHCs) and hearing thresholds up to two weeks following a mild noise exposure in mice. The mechanisms by which FOXO3 preserves cochlear cells and function are unknown. In this study, we analyzed the immediate effects of mild noise exposure on wild-type, Foxo3 heterozygous (Foxo3-het), and Foxo3-knockout (Foxo3-KO) mice to better understand Foxo3's roles in the mammalian cochlea. We used confocal and multiphoton microscopy to examine well-characterized components of noise-induced damage, including calcium regulators, oxidative stress, necrosis, and caspase-dependent and independent apoptosis. Lower immunoreactivity of the calcium buffer oncomodulin in Foxo3-KO OHCs correlated with cell loss beginning 4 hours post-noise exposure. Using immunohistochemistry, we identified parthanatos as the cell death pathway for OHCs. Oxidative stress response pathways were not significantly altered in FOXO3's absence. We used RNA sequencing to identify and RT-qPCR to confirm differentially expressed genes. We further investigated a gene downregulated in the unexposed Foxo3-KO mice that may contribute to OHC noise susceptibility. Glycerophosphodiester Phosphodiesterase Domain Containing 3 (GDPD3), a possible endogenous source of lysophosphatidic acid (LPA), has not previously been described in the cochlea. As LPA reduces OHC loss after severe noise exposure, we treated noise exposed Foxo3-KO mice with exogenous LPA. LPA treatment delayed immediate damage to OHCs but was insufficient to ultimately prevent their death or prevent hearing loss. These results suggest that FOXO3 acts prior to acoustic insult to maintain cochlear resilience, possibly through sustaining endogenous LPA levels.

Transcriptome Variant Analysis of Noise Susceptibility in C57BL/6J Mice

“…Prior to this, some animal studies have demonstrated that NOX3, FOXO3, NRF2, CX26, CRFR2, BHMT, A1AR, and MYH14 (Beaulac et al 2021; Fu et al 2016; Graham et al 2010; Honkura et al 2016; Lavinsky et al 2015; Partearroyo et al 2019; Vlajkovic et al 2017; Zhou et al 2016) knockout mice are more sensitive to noise than wild-type mice. These studies in knockout mice have shown that genetic defects in mice, which disturb specific paths and structures within the cochlea, make mice more sensitive to noise (Le et al 2017).…”

Development of a selective fluorescence-based enzyme assay for glycerophosphodiesterase family members GDE4 and GDE7

“…For ATX, compounds, such as FS-3, CPF-4, and TG-mTMP (18)(19)(20), have been reported as fluorescent substrates, and many inhibitors have been identified (21,22). In contrast, GDE4 has been suggested to be involved in retinitis pigmentosa (23), while GDE7 in fatty liver (24), cancer recurrence (25), and noise-induced hearing loss (26). However, no fluorescent substrates of GDE4 or GDE7 have been reported till date, and their enzymatic activities have been measured by TLC (12,14) and quantification of liberated choline (13),…”